The Ataxia Telangiectasia gene product, the ATM protein kinase, is required for cells to survive Ionizing Radiation (IR)-induced DNA damage. The signal transduction pathway which links IR-induced DNA damage to ATM activation is poorly characterized. We have identified a novel 114 amino-acid N-Terminal domain of ATM which is an essential component of this signal transduction pathway. Amino-acids 1-114 of ATM are required for cells to survive IR-induced DNA damage and for p53 binding to ATM. The hypothesis to be tested is that the N-terminal of ATM is required for ATM to bind to and phosphorylate key effector proteins, including p53, chk2, Brca1 and NBS. In specific aim 1, deletion analysis and site-directed mutagenesis will be used to identify the exact amino-acids which constitute this domain. ATM with this N-terminal domain inactivated will be expressed in Ataxia Telangiectasia cells. These cells will be exposed to IR, and the role of the N-Terminal regulatory domain of ATM in regulating cell survival, ATM kinase activity and association with p53 will be determined. In specific aim 2, we will determine the mechanism by which the N-Terminal of ATM regulates cell survival following exposure to IR. Cell lines expressing the ATM construct in which the N-terminal is inactivated will be exposed to IR and the ability of ATM to associate with and phosphorylate key ATM effectors, including the Brca1, p53, chk2 and NBS proteins, will be determined. The N- terminal of ATM will be expressed as a dominant negative inhibitor of ATM in human breast epithelial cells immortalized with human Telomerase. These cells will be used to examine ATM function in cells which are not compromised by viral or oncogene transformation. The identification of the exact position of this crucial N-terminal protein domain will elucidate the molecular basis by which ATM activates DNA damage induced signal-transduction and it will identify new molecular targets for ATM inhibition.